Note: Descriptions are shown in the official language in which they were submitted.
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ADRENERGIC AGONISTS FOR USE IN TREATING LIVER DAMAGE
The invention relates to liver damage, and to pharmaceutical compositions for
use in
treating, preventing or ameliorating liver damage or disease, especially acute
liver
damage. The invention is particularly, although not exclusively, concerned
with treating
or preventing liver damage caused by paracetamol (also known as acetaminophen)
poisoning. The invention also extends to methods of treating such conditions.
Paracetamol (Acetaminophen, APAP) overdose, either deliberate, through suicide
attempts, or unintentionally, because of consumption of multiple-drug
preparations
containing APAP, is a major public health problem worldwide because it causes
much
morbidity which frequently progresses to fulminant liver failure (FLF). This
is despite
the presence of N-Acetyl Cysteine (NAC) as an antidote, and Governmental
attempts to
reduce the non-prescription availability of APAP. FLF may result in death if a
suitable
liver for transplantation cannot be found, with about 200 such deaths per
year, in
England and Wales alone. Besides these deaths, there is the fiscal cost of
liver
transplantation and subsequent maintenance of these transplanted patients,
with total
such costs having being estimated worldwide at billions of dollars per year. A
shortage
of donor livers for transplantation as a treatment for liver disease,
including FLF, drives
the search to understand the factors that regulate liver regeneration.
There is therefore a need to provide an improved means of treating liver
disease or
damage. The inventors have surprisingly demonstrated that the activation of a-
adrenergic receptors and/or 3-adrenergic receptors, which are present on
hepatic
progenitor cells (stem cells, HPC), promotes the expansion of these stem cells
and can
therefore be used to treat liver damage.
Thus, in a first aspect of the invention, there is provided an adrenergic
receptor agonist,
for use in treating, preventing or ameliorating liver damage.
In a second aspect, there is provided a method of treating, ameliorating or
preventing
liver damage in a subject, the method comprising administering, to a subject
in need of
such treatment, a therapeutically effective amount of an adrenergic receptor
agonist.
Hepatic progenitor cells (HPC) are bi-potential liver resident stem cells that
can
differentiate into hepatocytes or bile duct cells. They are activated to
promote hepatic
regeneration and replace lost liver tissue after acute massive hepatocyte loss
or when
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mature hepatocyte replication is impaired, as in chronic liver inflammatory
conditions,
such as non-alcoholic steatohepatitis. Emerging evidence suggests that the
sympathetic
nervous system (SNS) may be involved in liver repair, either directly or
through effects
on liver cells, such as myofibroblastic hepatic stellate cells (HSC), which
are regulated
positively by the SNS. Also, it has previously been shown the al-adrenoceptor
antagonist, prazosin (PRZ), expanded liver progenitors and reduced injury in a
chronic
model of liver disease.
Therefore, in a further series of experiments with the P-adrenergic receptor
antagonist
propranolol (PRL), the inventor's starting hypothesis was that the homeostatic
effect of
SNS signalling on HPC expansion is inhibitory and that PRL would, as with PRZ,
expand HPC numbers and reduce liver injury. Initial results, under in vivo
conditions
simulating non-alcoholic steatohepatitis (NASH), showed emphatically that PRL,
like
PRZ, expanded the HPC population. However, PRL unlike PRZ, significantly
increased
biochemical and histological markers of liver injury and cell death.
Mechanistic studies
showed that PRL induced hepatocyte death, as evidenced by increased release of
ALT,
LDH, TNF-a and FAS ligand, through both the extrinsic and intrinsic apoptotic
pathways as judged by upregulation of FAS receptor, caspase-8 proteins, and
cytochrome C. These PRL results caused the inventors to modify their working
hypothesis and, as a result, they postulated that surprisingly, the basal
action of SNS
agonist signalling in liver injury may be to promote HPC expansion.
This modified hypothesis was supported by the finding that infusion of the SNS
agonists Norepinephrine (NE) and Isoprenaline (ISO) into spontaneously
steatohepatitic ob/ob mice induced increases in HPC number, and a parallel
reduction
in liver injury. Furthermore, in the complete absence of the SNS in mice
lacking
-/-
Dopamine P-hydroxylase (Dbh ) and which therefore cannot synthesize SNS
neurotransmitters, a diet inducing NASH led to a loss of the hepatomegaly and
expansion of HPC normally associated with this diet and observed in the
controls. This
reduction was reversed by infusion of ISO. Moreover, although HPC are
acknowledged
to play only a minor role in liver regeneration after a partial hepatectomy,
in the
absence of agents that inhibit replication of mature hepatocytes, the
inventors
surprisingly also observed a clear reduction in HPC numbers in the Dbh-i- mice
post
hepatectomy. These surprising results suggested unequivocally and for the
first time,
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that direct SNS agonist signalling is required to expand the HPC compartment
after
acute and chronic liver injury.
In support of the above there is evidence showing that the SNS regulates stem
cell
physiology in other organs, such that pharmacological manipulation of the SNS
has
been shown to modulate haematopoietic stem cell proliferation and egress.
Moreover,
adrenergic agents have been shown to induce proliferation of neuronal stem
cells and
embryonic stem cells have also been shown to respond to adrenergic
stimulation.
Given the clinical importance of APAP poisoning and evidence suggesting that
the SNS
regulates HPC and reduces liver injury, the inventors hypothesized that SNS
stimulation by ISO would expand the HPC population and reduce acute liver
injury
induced by APAP. They also sought to investigate the mechanisms through which
ISO
affected HPC. The results comprehensively show that HPC are markedly expanded
by
the SNS P-adrenoceptor agonist ISO through the 13-catenin-Wnt pathway and that
ISO
drastically reduces APAP induced injury. Since there is a possibility that ISO
may cause
abnormal cardiac rhythms in patients with acute APAP poisoning, the inventors
then
sought to determine if the al-adrenoceptor agonist phenylephrine, which may
induce
less abnormal rhythms, also caused an expansion of HPC with reduced liver
injury.
Accordingly, the adrenergic receptor agonist may be used for treating,
preventing or
ameliorating any kind of liver damage or failure. For example, the agonist may
be used
to treat fulminant liver failure (FLF). The liver damage which is treated may
be acute
liver damage. For example, the liver damage may have been caused by
administration
or consumption of a poison, for example paracetamol (i.e. APAP) or alcohol.
The liver
damage may have been caused by ingestion of Khat plant, which like APAP, may
also
cause acute liver failure (ALF).
Adrenergic receptors are metabotopic G-protein coupled receptors (GPCRs) that
are
activated by catecholamines, especially noradrenaline and adrenaline. These
receptors
are generally classified as either alpha(a)-adrenoceptors or beta([3)-
adrenoceptors.
Accordingly, in one embodiment, the adrenergic receptor agonist may be an a-
adrenergic receptor agonist. In another embodiment, the adrenergic receptor
agonist
may be a 3-adrenergic receptor agonist.
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The term "agonist" can mean a molecule that selectively binds to either the a-
or the 13-
adrenergic receptor to initiate the signal transduction reaction. Preferably,
the agonist
is operable, in use, to selectively activate the desired adrenergic receptor,
i.e. the
agonist activates the target adrenoceptor to a greater extent, or at lower
doses, than
other types of adrenergic receptors.
Alpha-adrenergic receptors may further be characterized as either a1-
adrenoceptors or
a2-adrenoceptors. Therefore, the adrenergic receptor agonist may be either an
a, or an
a2-adrenergic receptor agonist. Activation of alphai-adreonceptors promotes
the
activation of the G protein, Gq, which, in turn leads to the activation of the
phoshpolipase C signaling pathway, whereas activation of a2 adrenoceptors
promotes
the activation of the G protein, Gõ which in turn leads to the activation of
the adenylate
cyclase signaling pathway. Hence, a suitable a1-adrenergic receptor agonist
may be
selected from a group consisting of: Noradrenaline, Xylometazoline,
Phenylephrine,
and Methoxamine.
A preferred a1-adrenergic receptor agonist is Phenylephrine, as described in
Example 7.
A suitable a2-adrenergic receptor agonist may be selected from a group
consisting of:
Clonidine, Dexmedetomidine, Medetomidine, and Romifidine.
The skilled person will appreciate that a1-adrenoceptors may be further
subcategorized
as an-, a1,- or aid-adrenoceptors. a2-adrenoceptors may be further
subcategorized as
a2b- or a2cadrenoceptors.
Beta-adrenergic receptors may be further characterized, as betai-
adrenoceptors, beta2-
adrenoceptors or beta3-adrenoceptors. Therefore, the adrenergic receptor
agonist may
be a 13,-, a 132- or a 133-adrenergic receptor agonist. However, in some
embodiments, the
agonist may not be a 133-adrenergic receptor agonist. Stimulation of either of
the three
13-adrenergic receptors promotes the activation of the G protein, G, which in
turn leads
to the activation of the adenylate cyclase signaling pathway. A suitable 131-
adrenergic
receptor agonist may be selected from a group consisting of: Dobutamine,
Isoprenaline,
and Noradrenaline. A preferred 131-adrenergic receptor agonist is
Isoprenaline, as
described in the Examples.
A suitable 132-adrenergic receptor agonist may be selected from a group
consisting of:
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Isoprenaline and Salbutamol. As described in the Examples, these agonists will
be
useful in the treatment of acute liver disease/damage.
In some embodiments of the invention, it may be desirable to administer an a-
adrenoceptor agonist and a 3-adrenoceptor agonist simultaneously. For example,
an ai-
adrenergic receptor agonist such as Noradrenaline, Xylometazoline,
Phenylephrine, or
Methoxamine may be administered together with a (31-adrenergic receptor
agonist such
as Dobutamine, Isoprenaline, and Noradrenaline. Preferably, Phenylephrine is
administered with Isoprenaline.
Classification of a-adrenoceptors and P-adrenoceptors, and their subtypes, may
be
achieved by comparing the potency of the catecholamines, isoprenaline,
adrenaline and
noradrenaline at each of these receptors, and possibly also by determining the
type of
intracellular signaling pathway which is activated by the action of an agonist
at the
receptor.
Adrenergic receptor agonists used according to the invention may achieve their
functional effect through promoting the expansion of hepatic progenitor cells
(HPC's).
Although not wishing to be bound by any hypothesis, the inventors believe that
adrenoceptor agonists promote expansion/proliferation of hepatic progenitor
cells
through activation of the HPC Wnt pathway, which leads to the expression of
various
Wnts. Wnts are a family of signaling proteins which pass signals from
receptors found
on the surface of cells to their nuclei to regulate gene expression.
Accordingly, the agonist may be operable in use to enhance HPC expansion,
preferably
by activating the Wnt pathway.
Therefore, in a third aspect, there is provided an adrenergic receptor
agonist, for use in
inducing the expression of Wnt by hepatic progenitor cells.
Preferably, expression of Wnt 1, 3a, 6 or ma may be induced by the agonist
compared
to the level of expression in the absence of the agonist.
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The term "expression" can relate to the detection of a Wnt protein in any
compartment
of the cell (e.g. in the nucleus, cytosol, the Endoplasmic Reticulum or the
Golgi
apparatus); or detection of the mRNA encoding a Wnt.
It will be appreciated that adrenoceptor agonists according to the invention
may be
used in a medicament, which may be used in a monotherapy, i.e. use of only an
adrenoceptor agonist (e.g. an antibody or a catecholamine) for treating,
ameliorating,
or preventing acute liver damage/disease. Alternatively, adrenoceptor agonists
according to the invention may be used as an adjunct to, or in combination
with, known
therapies for treating, ameliorating, or preventing acute liver
damage/disease. For
example, adrenoceptor agonists of the invention may be used in combination
with
known agents for treating acute liver damage/disease, such N-Acetyl Cysteine
etc.
The adrenoceptor agonists according to the invention may be combined in
compositions having a number of different forms depending, in particular, on
the
manner in which the composition is to be used. Thus, for example, the
composition
may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel,
hydrogel,
aerosol, spray, micellar solution, transdermal patch, liposome suspension, or
any other
suitable form that may be administered to a person or animal in need of
treatment. It
will be appreciated that the vehicle of medicaments according to the invention
should
be one which is well-tolerated by the subject to whom it is given.
The composition may comprise liver-targeting means, arranged, in use, to
target the
adrenoceptor agonist at least adjacent the liver. For example, the
adrenoceptor agonist
may be formulated within a liposome or liposome suspension, which liposome
comprises a ligand which targets the liver. Advantageously, such liver
targeting
significantly improves delivery of the active agent to the treatment site
increasing
efficacy.
Medicaments comprising adrenoceptor agonists according to the invention may be
used in a number of ways. For instance, oral administration may be required,
in which
case the adrenoceptor agonists may be contained within a composition that may,
for
example, be ingested orally in the form of a tablet, capsule or liquid.
Compositions
comprising adrenoceptor agonists of the invention may be administered by
inhalation
(e.g. intranasally). Compositions may also be formulated for topical use. For
instance,
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creams or ointments may be applied to the skin, for example, adjacent the
treatment
site, e.g. the liver.
Adrenoceptor agonists according to the invention may also be incorporated
within a
slow- or delayed-release device. Such devices may, for example, be inserted on
or under
the skin, and the medicament may be released over weeks or even months. The
device
may be located at least adjacent the treatment site. Such devices may be
particularly
advantageous when long-term treatment with adrenoceptor agonists used
according to
the invention is required and which would normally require frequent
administration
(e.g. at least daily injection).
In a preferred embodiment, adrenoceptor agonists and compositions according to
the
invention may be administered to a subject by injection into the blood stream
or
directly into a site requiring treatment. Injections may be intravenous (bolus
or
infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or
infusion).
It will be appreciated that the amount of the adrenoceptor agonist that is
required is
determined by its biological activity and bioavailability, which in turn
depends on the
mode of administration, the physiochemical properties of the adrenoceptor
agonist and
whether it is being used as a monotherapy or in a combined therapy. The
frequency of
administration will also be influenced by the half-life of the adrenoceptor
agonist
within the subject being treated. Optimal dosages to be administered may be
determined by those skilled in the art, and will vary with the particular
adrenoceptor
agonist in use, the strength of the pharmaceutical composition, the mode of
administration, and the advancement of the disease being treated. Additional
factors
depending on the particular subject being treated will result in a need to
adjust dosages,
including subject age, weight, gender, diet, and time of administration.
Generally, a daily dose of between o.olvtg/kg of body weight and o.5g/kg of
body
weight of the adrenoceptor agonist according to the invention may be used for
treating,
ameliorating, or preventing liver damage/disease, depending upon which
adrenoceptor
agonist is used, e.g. catecholamine or antibody. More preferably, the daily
dose of the
adrenoceptor agonist is between o.oimg/kg of body weight and 500mg/kg of body
weight, more preferably between o.img/kg and 200mg/kg body weight, and most
preferably between approximately img/kg and ioomg/kg body weight.
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As discussed in the examples, particularly Examples 6 and 7, the adrenoceptor
agonist
may be administered before, during or after onset of acute liver
disease/damage. For
example, the agonist may be administered immediately after a subject has
ingested a
toxic amount of paracetamol. Daily doses may be given as a single
administration (e.g.
a single daily injection). Alternatively, the adrenoceptor agonist may require
administration twice or more times during a day. As an example, adrenoceptor
agonists
may be administered as two (or more depending upon the severity of the disease
being
treated) daily doses of between 25mg and 7000 mg (i.e. assuming a body weight
of 70
kg). A patient receiving treatment may take a first dose upon waking and then
a second
dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals
thereafter.
Alternatively, a slow release device may be used to provide optimal doses of
adrenoceptor agonist according to the invention to a patient without the need
to
administer repeated doses.
In another embodiment, the adrenoceptor agonist may be administered before the
onset of liver damage. For example, in cases where a subject is undergoing
clinical trials
or being treated with a drug which is known to, or likely to, cause acute
liver damage
(for example an anticancer drug), then it may be advantageous to protect the
liver by
per-administering the adrenoceptor agonist of the invention.
Known procedures, such as those conventionally employed by the pharmaceutical
industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to
form specific
formulations comprising the adrenoceptor agonist according to the invention
and
precise therapeutic regimes (such as daily doses of the adrenoceptor agonist
and the
frequency of administration). The inventors believe that they are the first to
describe a
pharmaceutical composition for treating acute liver disease/damage, based on
the use
of the agonist of the invention.
Hence, in a fourth aspect of the invention, there is provided a liver damage
treatment
composition, comprising an adrenergic receptor agonist and a pharmaceutically
acceptable vehicle.
Liver damage or disease which may be treated with the composition may be
acute. In
addition, the liver disease may be caused by a variety of factors, which can
include
paracetamol or Acetaminophen (APAP) overdose, alcoholism, or other diseases,
such as
Malaria. The agonist may comprise an a- or a P-adrenergic receptor agonist. In
one
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embodiment, the agonist may be either an a, or an a2-adrenergic receptor
agonist. A
suitable a1-adrenergic receptor agonist may be selected from a group
consisting of:
Noradrenaline, Xylometazoline, Phenylephrine, and Methoxamine. Preferably, the
agonist is Phenylephrine. A suitable a2-adrenergic receptor agonist may be
selected
from a group consisting of: Clonidine, Dexmedetomidine, Medetomidine, and
Romifidine. In another embodiment, the agonist may be a 13,-, a 132- or a 133-
adrenergic
receptor agonist. A suitable 131-adrenergic receptor agonist may be selected
from a
group consisting of: Dobutamine, Isoprenaline, and Noradrenaline. Preferably,
the
agonist is Isoprenaline. A suitable 131-adrenergic receptor agonist may be
selected from
a group consisting of: Isoprenaline and Salbutamol.
The invention also provides in a fifth aspect, a process for making the
composition
according to the fourth aspect, the process comprising contacting a
therapeutically
effective amount of an adrenergic receptor agonist and a pharmaceutically
acceptable
vehicle.
A "subject" may be a vertebrate, mammal, or domestic animal. Hence,
compositions
and medicaments according to the invention may be used to treat any mammal,
for
example livestock (e.g. a horse), pets, or may be used in other veterinary
applications.
Most preferably, however, the subject is a human being.
A "therapeutically effective amount" of the adrenoceptor agonist is any amount
which,
when administered to a subject, is the amount of medicament or drug that is
needed to
treat liver disease/damage or produce the desired effect.
For example, the therapeutically effective amount of adrenergic receptor
agonist used
may be from about 0.01 mg to about 800 mg, and preferably from about 0.01 mg
to
about 500 mg. It is preferred that the amount of adrenoceptor agonist is an
amount
from about 0.1 mg to about 250 mg, and most preferably from about 0.1 mg to
about 20
mg.
A "pharmaceutically acceptable vehicle" as referred to herein, is any known
compound
or combination of known compounds that are known to those skilled in the art
to be
useful in formulating pharmaceutical compositions.
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In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and
the
composition may be in the form of a powder or tablet. A solid pharmaceutically
acceptable vehicle may include one or more substances which may also act as
flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers,
glidants,
compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or
tablet-
disintegrating agents. The vehicle may also be an encapsulating material. In
powders,
the vehicle is a finely divided solid that is in admixture with the finely
divided active
agents according to the invention. In tablets, the active agent (e.g. the
adrenoceptor
agonist) may be mixed with a vehicle having the necessary compression
properties in
suitable proportions and compacted in the shape and size desired. The powders
and
tablets preferably contain up to 99% of the active agents. Suitable solid
vehicles include,
for example calcium phosphate, magnesium stearate, talc, sugars, lactose,
dextrin,
starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion
exchange
resins. In another embodiment, the pharmaceutical vehicle may be a gel and the
composition may be in the form of a cream or the like.
However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical
composition is in the form of a solution. Liquid vehicles are used in
preparing solutions,
suspensions, emulsions, syrups, elixirs and pressurized compositions. The
adrenoceptor agonist according to the invention may be dissolved or suspended
in a
pharmaceutically acceptable liquid vehicle such as water, an organic solvent,
a mixture
of both or pharmaceutically acceptable oils or fats. The liquid vehicle can
contain other
suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers,
preservatives, sweeteners, flavouring agents, suspending agents, thickening
agents,
colours, viscosity regulators, stabilizers or osmo-regulators. Suitable
examples of liquid
vehicles for oral and parenteral administration include water (partially
containing
additives as above, e.g. cellulose derivatives, preferably sodium
carboxymethyl cellulose
solution), alcohols (including monohydric alcohols and polyhydric alcohols,
e.g.
glycols) and their derivatives, and oils (e.g. fractionated coconut oil and
arachis oil). For
parenteral administration, the vehicle can also be an oily ester such as ethyl
oleate and
isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form
compositions
for parenteral administration. The liquid vehicle for pressurized compositions
can be a
halogenated hydrocarbon or other pharmaceutically acceptable propellant.
Liquid pharmaceutical compositions, which are sterile solutions or
suspensions, can be
utilized by, for example, intramuscular, intrathecal, epidural,
intraperitoneal,
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intravenous and particularly subcutaneous injection. The adrenoceptor agonist
may be
prepared as a sterile solid composition that may be dissolved or suspended at
the time
of administration using sterile water, saline, or other appropriate sterile
injectable
medium.
The adrenoceptor agonist and pharmaceutical compositions of the invention may
be
administered orally in the form of a sterile solution or suspension containing
other
solutes or suspending agents (for example, enough saline or glucose to make
the
solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate,
polysorbate 80 (oleate
esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and
the like.
The adrenoceptor agonists according to the invention can also be administered
orally
either in liquid or solid composition form. Compositions suitable for oral
administration include solid forms, such as pills, capsules, granules,
tablets, and
powders, and liquid forms, such as solutions, syrups, elixirs, and
suspensions. Forms
useful for parenteral administration include sterile solutions, emulsions, and
suspensions.
All of the features described herein (including any accompanying claims,
abstract and
drawings), and/or all of the steps of any method or process so disclosed, may
be
combined with any of the above aspects in any combination, except combinations
where at least some of such features and/or steps are mutually exclusive.
For a better understanding of the invention, and to show how embodiments of
the same
may be carried into effect, reference will now be made, by way of example, to
the
accompanying Figures, in which:-
Figure IA shows the mean number of CI(1.9 positive HPCs in the liver of mice
control
Dbh+/ , Dbh / , and Dbh / mice infused with Isoprenaline (Dbh / +ISO) at 20
mg/kg/day to induce activation of the SNS. Data are mean s.e.m, n= 5 mice
per
group. *p < 0.05 in Dbh / mice compared to control mice and #p < 0.05 in Dbh /
+ ISO
compared to Dbh / (one-way ANOVA with Tukey's post hoc test);
Figure iB shows the results of a duplex PCR performed on isolated EpCAM+ cells
(EpCAM+ cells) and EpCAM depleted non-parenchyma cells (EpCAM-cells) from
normal mouse liver. Total liver extract served as control;
Figure iC are representative flow cytometry plots of side population (SP)
cells in total
NPC isolated from normal mice liver. The same samples treated with verapamil
which
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inhibit the function of the ABC transporter lost the SP population. EpCAM
positive cells
were highly enriched in the SP cells. Inset number indicates percentage of
positive cells
in total NPC;
Figure 1D shows adrenoceptor mRNA expression of isolated EpCAM+ cells and the
liver progenitor cell line (6o3B cells) using RT-PCR;
Figure 1E (Left panel) are the results of a cell proliferation assay which
show the fold-
increase in the number of 6o3B cells at different doses of isoprenaline (loopM
¨
io[tM). Results are expressed as fold change s.e.m from 6 biological
replicates relative
to control (basal medium). **p<o.00i compared to basal medium control (one-way
ANOVA with Tukey's post hoc test);
Figure iE (Right panel) are the results of a cell proliferation assay which
show the fold-
increase in the number of 6o3B cells treated with basal medium (basal) as
control,
lo[IM of isoprenaline, and lo[IM of isoprenaline (ISO) after pre-treatment
with io[IM
of Propranolol (ISO+PRL). Results are expressed as fold change s.em, relative
to basal
from 3 biological replicates; *p<o.o5 compared to basal; #p<o.05 compared to
ISO;
Figure iF shows the percentage of EpCAM+ cells (determined using flow
cytometry),
the number of CI(19 cells (determined using Immunohistochemistry) and the
number
of EpCAM cells in (the livers of mice) mice which received either a control
treatment
(Con) or isoprenaline (Iso) at a dose of 2.5mg/kg. Control mice received PBS
vehicle
(Con). Data are representative from 2 independent experiments. Data are mean
s.e.m. (n=4 per group);
Figure iG are representative images of HPCs detected in mice livers using
immunohisotchemistry. Mice received either a control treatment (Con) or
isoprenaline
(ISO);
Figure 2A is a representative western blot and densitometric analyses showing
indicated protein expression in 6o3B cells treated with either io[IM
isoprenaline (ISO)
or basal media (Con). An antibody to 13-actin was used as a loading control.
Data are
mean s.e.m, n= 3;
Figure 2B are immunoflurorescent immunocytochemistry images of 6o3B cells
treated
with isoprenaline showing cell membrane localisation of 13-catenin (left) and
nuclear
localization of 13-catenin (right). Nuclei were stained with dapi (blue);
Figure 2C is a proliferation assay showing the fold-increase in the number of
6o3B cells
stimulated with lo[IM isoprenaline (ISO) or io[IM ISO in the presence (or
absence) of
i[IM of the specific Wnt/13 catenin inhibitors XAV939 (XAV) and PNU-74654
(PNU).
Data are mean s.e.m. (n= 3); **p<o.00i compared to basal medium; #p<o.o5
compared to ISO;
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Figure 2D shows the level of Wnt ligand (Wnt 1, 3a & 6) mRNA expression,
assessed by
real- time PCR, in 6o3B cells treated with io[IM of isoprenaline or control
(Con). Data
are mean s.e.m. (n= 4), *p<0.05;
Figure 2E are the results of a CCK8 proliferation assay which show the effect
of the Wnt
antagonist (recombinant Dkk 1, 0.1 pg/m1) on isoprenaline (1o[tM)-stimulated
6o3B
cells. Data are mean s.e.m (n= 3);
Figure 2F are immunohistochemical stains for active 13-catenin in a mouse
liver 24
hours after administration of vehicle (Con) or Isoprenaline (ISO). Left panel
= lower
magnification; Right panel = higher magnification;
Figure 3A is a graph showing that ALT (top ) and % necrosis (bottom) are
cumulative
for all animals either injected with vehicle, isoprenaline (2.5mg/kg), APAP
(375mg/kg)
or APAP with subsequent administration of isoprenaline (A+I) at 24h after
first
administration; n=11 to 22 per group;
Figure 3B is a representative histological image of mice liver 24 hours after
injection
with either vehicle (control), isoprenaline (2.5mg/kg), APAP (375mg/kg) or
APAP with
subsequent administration of isoprenaline (A+I); left hand panel in each
figure is the
lower magnification and the right hand panel the higher magnification;
Figure 3C shows the ALT 3h of mice after APAP administration. Data are mean
s.e.m,
n=4/group; *p=o.o5, by 2- tailed unpaired t- test;
Figure 3D is the flow cytometric analysis of CD45-/EpCA1VI+ve cells in non-
arenchymal
cell fraction. Data are mean s.e.m, n=4/group; *p<o.o5, 'p<o.00l;
Figure 3E is the immunohisotchemical analyses of progenitor cells using EpCAM
(left)
and CK19 (right). Data are mean numbers of EpCAM and CK19 +ve cells per portal
tract s.e.m (n= 5/group);
Figure 3F shows CK19 positive cell density confined to small portal tracts;
'p<o.00l;
Figure 3G shows liver injury judged by ALT elevation (left) and HPC number
determined by CK19 positive cells density (right) in mice treated with APAP
alone or
APAP with subsequent administration of PRL (lomg/kg). Data are mean s.e.m,
n=4
each, *p<o.o5, ns = not significant;
In Figures 3A-3G, Con = vehicle treated, APAP = 375mg/kg of APAP and
subsequent
vehicle treatment, A+ISO = 375mg/kg of APAP and subsequent 2.5mg/kg of ISO
treatment;
Figure 4A is a representative western blot (upper) and densitometric (lower)
analyses
of 13-catenin in the livers of mice treated with APAP or APAP + ISO. ISO was
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administered 1 hour after APAP and the livers were harvested 24h after APAP
initial
administration; n=4 per group. *p<0.05;
Figure 4B are representative micrographs of immunohistochemiacal staining of
active
13-catenin in the livers of mice treated with APAP or APAP/ISO (A+I) 24h after
initial
administration as in (a) above. Upper panel = lower magnification, Lower
panel=
higher magnification;
Figure 4C shows wnt ligand expression in total liver 24h after initial
administration
were analyzed in vehicle injected (Con), APAP injected (APAP), and APAP with
subsequent ISO injected (A+I) mice. Data are mean s.e.m, n=4 each. *p<0.05,
**p<0.001;
Figure 4D shows the level of wnt ligand expression in EpCAM+ve cells, the
EpCAM
depleted non-parenchymal fraction, and hepatocytes isolated from mice livers
treated
with APAP and ISO 24h after initial APAP administration;
Figure 4E are the results of a LDH cell cytotoxicity assay. Isolated
hepatocytes from
normal mice liver were treated with lomM APAP or control medium in the
presence or
absence of ISO (loopM ¨ ioluM), 20mM of N-acethylsysteine (NAC), loong/m1
recombinant mouse Wnt3a, Wnt3a with recombinant mouse Dkki (0.1 pg/m1), 6o3B
conditioned medium from cells stimulated with ISO (CM) and CM with Dkki. Each
bar
represents replicates from 6 wells of the same treatment. Results are
expressed as fold
change s.e.m. relative to triton X treated hepatocyte as controls. *p<0.05
compared to
APAP alone;
Figure 5A shows the affect of recombinant TWEAK, 0.04 [tg/g on liver injury,
assessed
using ALT;
Figure 5B shows the affect of recombinant TVVEAK, 0.04 [tg/g on CK19+ve HPC
cell
numbers;
Figure 5C are representative images of immunohistochemical staining with CK1.9
(DAB
chromogen, brown), upper panels; and CK19 with Ki67 (AEC chromogen, red),
double
staining (middle panels) and NFKB p65 immunostaining (lower panels). Insert =
higher
magnification, arrow head indicates positive staining of ki67 in CK 19 +ve
HPCs. White
arrow head indicates nuclear localization of NFKb p65 in periportal ductular
cells;
Figure 5D shows the experimental design of the TWEAK study;
Figure 5E shows the % necrosis (left) and serum ALT (right) from mice treated
with
APAP and TWEAK pre-treated mice with subsequent APAP administration. Data are
mean s.e.m, n=4 each. *p<0.05, 24h after initial APAP administration;
Figure 5F are representative histological images of APAP and TWEAK/APAP mice
livers;
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Figure 5G shows the experimental design of EpCAM positive cell administration.
Mice
were administered APAP. One and half hours later they given EpCAM +ve cells,
or
EpCAM +ve cells with/without DKKi, EpCAM depleted NPC or vehicle. EpCAM +ve
cells were isolated from APAP + ISO treated mice;
Figure 5H shows the production of the liver injury marker, ALT, 24h after APAP
administration. Serum ALT (left) and % necrosis (right) were analyzed in APAP
+
vehicle (APAP), APAP and EpCAM depleted non-parenchymal cells (A+NPC), APAP +
EpCAM +ve (A+Epc), and APAP + EpCAM +ve (A+Epc+DKI(1). Data are mean s.e.m,
n=4/group. *p<0.05;
Figure 6A shows the experimental design of the study used to obtain the
results of
Figure 6B;
Figure 6B shows the ALT of mice which received APAP and 1 or 3hrs later were
received NAC. An alternative batch of mice was treated with APAP followed by
ISO 3hrs
after initial APAP;
Figure 6C shows the immunohistochemical staining of mice which were used in
Figure
6B;
Figure 7a shows the ALT of mice treated with APAP followed 1 hr later by
phenylephrine (PE, 3mg/kg or lomg/kg) and sacrifice 24hr5 after APAP
administration; and
Figure 7b shows the affect of PE (lopM to io[tM) on the proliferation of 603B
cells.
Data are mean s.e.m. (n= 3). **p<0.01 compared to control (Con).
Figure 8 shows that ISO induces 13 catenin activation on HPCs in vivo.
iorng/kg of ISO treated liver were subjected to analysis of active P-catenin
(red)
expression in pan-cytokeratin positive HPCs (green). Positive P-catenin
nuclear
staining is as shown on HPCs (yellow).
Materials & Methods
Animals
Male C57BL/6j mice with a mean weight 25 to 3og were from our Biological
Services
colony. Male dopamine P-hydroxylase deficient (Dbh-/-) and Dbh+/- mice (30-40
weeks) were also from our colony as previously described (Oben, J.A., et al.,
2004). All
animals were housed in an environmentally controlled room with 12-h light/dark
cycle
and allowed free access to food and water. All animals were treated in
accordance with
The Animals (Scientific Procedures) Act, UK, 1986 guidelines.
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Materials
Culture medium was obtained from Invitrogen. All other chemicals were from
Sigma
unless otherwise stated.
Cell line
Immature murine cholangiocyte cell line (6o3B cells) were a kind gift from
Professor
Diehl.
Animal experiments
All mice are fasted overnight before APAP administration. APAP was dissolved
in warm
phosphate buffered saline (PBS) and administered intra-peritoneally (IP) with
APAP at
a dose of 375mg/kg, 500mg/kg or PBS as control. One hour after APAP injection,
either
Propranolol in water (4mg/kg), Isoproterenol (ISO) in water (2.5mg/kg) or
water were
administered IP. 24 hours after APAP treatment, mice were sacrificed with
carbon
dioxide. Dbh-/- mice were administered ISO as previously described
(Mackintosh, C.A.,
et al. 2000). Mouse recombinant TWEAK (R&D systems) was administered IP at
0.04
[tg/g body weight.
Cell isolation
Hepatocytes were isolated as previously reported (Schwabe, R.F., et al.,
2001). Hepatic
stellate cells, Kupffer cell and hepatic sinusoidal lining cell were extracted
by optiprep
gradient and subsequent selective adherence method as previously reported
(Oben,
J.A., et al., 2004; Li, Z., et al. 2002; and Williams, J.M., et al. 2010).
Purity of HSC,
KC, SEC was assessed by immunocytochemistry using GFAP, aSMA, F4/8o and vWF
antibody and revealed 98%, 92%, 87% purity respectively. EpCAM+ cell were
isolated
by BD Magnet according to the manufacturer's instructions.
Assessment of liver injury
The degree of liver injury was assessed by histology and serum ALT. All liver
sections
were stained with haematoxylin and eosin (H&E) and scanned by NanoZoomer
(Hamamatsu, Japan). Necrotic area was measured and expressed as a percentage
of
necrotic tissue in whole area of liver section using NDP.view (Hamamatsu,
Japan).
Cell culture experiments
6o3B cell were cultured as previously described (Omenetti, A., et al 2009).
For
proliferation assay, FBS was reduced to 1% and used as a basal state. LSEC
were
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cultured on collagen coated plate and other liver cell fractions were cultured
on normal
dish using RPMI-1640 containing io%FBS. Primary hepatocytes were cultured on
either collagen coated 96 well plate or 60mm dish using Williams E medium
supplemented with 10% FBS, insulin-trasferrin- selenium G cocktail and monM
dexamethasone. After 4 hours plating, cells were washed and replaced with
basic media
containing the reagents and incubated for a further 2 hours. After 2 hour of
reagents
treatment, APAP containing media adjusted to lomM of final concentration was
added.
Concentration of the drugs we used in this experiment was decided on the basis
of
preliminary experiments (data not shown).
Proliferation assay
CCk-8 assay
Cell proliferation assay was performed using the Cell counting Kit-8 (CCK-8)
according
to the manufacturer's protocol.
Direct cell counts
To further confirm the CCK-8 assay we also directly counted the cell numbers
in some
experiments. Adherent cells were treated with 0.25% trypsin solution
containing 0.02%
EGTA in Ca2+ and Mg2+ free phosphate-buffered saline at 37 C for 5 min, and
the
viable cell number and dead cell number was determined using Nucleocounter
(Chemometec).
Cytotoxic assay
LDH released from cells were assessed by LDH assay kit (Cayman) with o.1%
Triton X
treated cells as positive controls.
Immunohistochemistry (IHC)
Formalin-fixed paraffin-embedded tissue were cut at 4[Im onto glass slides
coated with
poly-l-lysine. For chromogenic IHC, antibody binding was visualized using the
ImmPRESS Peroxidase Polymer Detection Reagents (Vector lab, UK). For double
chromogenic IHC, microwave heat treatment in citric based solution (Vector
lab, UK)
were applied after the first color development. For immunofluorescence IHC or
immunocytochemistry, Alexa Fluor 555 and Alexa Fluor 488 conjugated secondary
antibody were used. Nuclei were stained with DAPI (Vector). All images were
captured
using a Nikon Eclipse e600 microscope and camera (DX1\41200F) and acquired
with
NIS-Elements Advance software (Nikon). HPC numbers were counted by an expert
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liver pathologist unaware of the identity of the groups as previously
described (Oben,
J.A., et al. 2003).
PCR and semi-quantitative real time PCR
RNA was isolated using TRIzol (Invitrogen), according to the manufacturer's
instructions. cDNA was synthesized with the Qiagen QuantiTect Reverse
Transcription
kit (Qiagen).
Duplicate PCR reactions were performed with multiplex PCR kit (Qiagen) using
mixed
primer (GAPDH and target primer). Semi-quantitative real time PCR was done
with
Rotor-Gene 3000 (Corbett Robotics) and QuantiFast SYBR Green PCR kit (Qiagen).
All
real-time PCR reactions were performed in triplicate with GAPDH as an internal
control. Target gene levels in treated samples are presented as a ratio to
levels detected
in corresponding control samples, according to the AACt method.
Western blotting
Western blotting was performed as described (Soeda, J et al., 2012). Western
blots
shown are representative of 2 or 3 independent repeats. Semi-quantitative
analysis of
western blots by densitometry was carried out using LabWorks 4.6 software
(UVP,
USA).
Flow cytometry
Total NPC were extracted and analysed as previously described (Okabe, M., et
al. 2009;
Yovchev, M.I., et al. 2008; and Lin, K.K. and M.A. Goodell, 2011). Hoechst3332
staining was performed as described (Lin, K.K. and M.A. Goodell, 2011; and
Goodell,
M.A., et al., 1996) with minor modifications. Briefly, total NPC were adjusted
to 106
cells/ml in pre-warmed RPMI complete media (io%FBS, P/S, galutamate),
incubated
for 90 minutes at 37 degree with 5ug/m1 of Hoechst with verapamil as (5ouM)
control.
Samples were then washed with ice-cold PBS and incubated with Fc blocker
(Cd16/32
mouse monoclonal antibody:BD) and stained with PE conjugated EpCAM (Biolgend)
and Alexa ¨fluor 700 conjugated CD45. Data were analyzed by FlowJo software
(version and company).
Statistical analyses
All data were expressed as mean s.e.m. and means were compared by the
Student's t-
test or ANOVA as appropriate. Sample size per group, n = />3 per group.
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Example 1 - The SNS Regulates Hepatic Progenitor Cell (HPC) Expansion
To determine if the SNS regulates HPC expansion, it was first confirmed
whether Dbh 1
(Dopamine fl-hydroxylase) mice, which are genetically deficient in the SNS
neurotransmitters norepinephrine (NE) and epinephrine, have a significantly
attenuated HPC population compared to their heterozygote controls. HPC
populations
were enumerated by the immunohistochemical presence of CK-19. As shown in
Figure
ia, treatment with isoprenaline (ISO), a non-specific [3-adrenoceptor agonist,
significantly recovered HPC numbers in Dbh 1 mice.
To confirm the expression of adrenoceptors on HPCs, EpCAM+ve cells were
isolated
from the livers of control C57BL mice. Expression of EpCAM (epithelial cell
adhesion
molecule) has been shown to be a reliable marker of HPCs in mice (Schmelzer,
E., et al
2007; Tanaka, M., et al. 2009; Okabe, M., et al. 2009; Yovchev, M.I., et al
2007). These
EpCAM+ve cells expressed other known HPC markers, for example CI(19, Sox9,
TROP2, and Oct4, as shown in Figure ib. The EpCAM+ve cells also showed Hoechst
33342 extruding properties, i.e. they were side population (SP) cells, as
shown in Figure
ic. Moreover, these HPCs, expressed aib-, aic-, a2a-, a2b-, (12C-, plus 131-
and 132-
adrenergic receptor subtypes at the mRNA level, as show in Figure id.
The above results were corroborated by double immunofluorescent staining with
pan-
cytokeratin (another accepted HPC marker (Yin, L., et al 2002)) and 131 and
132
adrenoceptor. This confirmed that 131 and 132 adrenoceptors are expressed on
HPCs at
the protein level. Therefore, there is an association between the expansion of
HPC
populations and the SNS, mediated via adrenoceptor.
To further delineate the role of adrenergic stimulation in HPC proliferation,
the 6o3B
cell line was used. 6o3B cells, like HPCs, are derived from the terminal
branches of the
biliary tree (Ueno, Y., et al 2003; Omenetti, A., et al 2007). As shown in
Figure id,
6o3B cells possess the same adrenoceptor profile as isolated EpCAM+ve cells.
This
finding validated their further use in this study. Treatment with ISO induced
6o3B cell
proliferation and their pre-treatment with the 13-adrenoceptor antagonist,
propranolol
(PRL), inhibited ISO induced proliferation, as shown in Figure ie.
Surprisingly, ISO
also increased the number of the HPC cells in normal C57BL/6J mice, as
determined by
expression of EpCAM (flow cytometry) and CI(19 (immunohistochemistry), as
shown in
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Figure if and Figure ig. Therefore, these findings suggested direct expansion
of HPCs
by ISO in murine liver.
Example 2- fl-adrenoceptor stimulation activates the canonical Wnt pathway on
HPCs
To elucidate the molecular pathway through which stimulation of P-
adrenoceptors
induces proliferation of HPCs, the canonical Wnt pathway was investigated. As
shown
in Figure 2a, total 13-catenin expression was significantly increased in ISO
treated 6o3B
cells. Expression of dephophorelated 13-catenin (activated (3-catenin) and
cyclin Di
which is a known 13-catenin target gene were also significantly upregulated in
ISO
treated 6o3B cells. Furthermore, immunofluorescence cytochemistry showed
accumulation of 13-catenin in the nuclei of ISO treated 6o3B cells, as shown
in Figure
2b. This data suggests that ISO treatment activated the canonical Wnt pathway
in 6o3B
cells.
To further elucidate the molecular pathway through which stimulation of 13-
adrenoceptors induces proliferation of HPCs, the effect of 13-catenin specific
inhibitors
were used in proliferation assays with 6o3B cells. As shown in Figure 2c, ISO-
induced
proliferation was partially but significantly inhibited by 13-catenin specific
inhibitors.
This indicates that the effect of ISO on 6o3B proliferation is partly mediated
by 13-
catenin. ISO treatment also significantly increased Wnti, 3a, 6 and ma mRNA
expression in 6o3B cells, as shown in Figure 2d. These Wnt ligands are known
to
activate the canonical Wnt pathway (Koch, S., et al 2on) and thus suggest that
ISO-
induced 6o3B proliferation is partly autocrine in nature.
To confirm the findings of Example 6 (above), the Wnt antagonist DKKi (Koch,
S., et al
2on) was used in the presence of ISO. Figure 2e shows that there was a trend
towards
statistical difference between the proliferation of ISO-treated and ISO plus
DKKi-
treated 6o3B cells.
The effect of 13-adrenoceptor stimulation on the canonical Wnt pathway was
studied
further in vivo. As shown in Figure 2f, mice treated with ISO showed
upregulation of
Wnt6 mRNA in total liver at 24h after injection and strong P-catenin
immunoreactivity
on periportal ductular cell detected by immunohistochemistry. Double
immunoflurorescence confirmed these cells were HPCs (see Figure 8). These
results
indicate that ISO treatment also activates the canonical Wnt pathway on HPC in
vivo.
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Example 3 - ISO protects against APAP-induced liver injury and enhances HPCs
expansion
To determine whether above-mentioned findings have any relevance with respect
to
liver disease, an APAP induced liver injury model which results in massive
hepatic
necrosis and progenitor cell proliferation was used (Williams, C.D. et al
2011; Kofman,
A.V., et al 2005).
Mice were initially administered APAP at 500mg/kg intraperitoneally. This
resulted in
a significant number of deaths, and was reduced by ISO treatment. Therefore,
the dose
of APAP was reduced to 375mg/kg. ih after administration with APAP, mice were
treated with either ISO or PBS vehicle. As shown in Figures 3a and 3h, APAP
treatment
induced massive hepatic necrosis as judged by histology and ALT 24h after APAP
treatment. ISO treatment significantly reduce the ALT (3332 462.9 vs. 674.1
173
IU/L, p<o.000l) and hepatic necrosis (35.25 3.745 vs. 18.48 1.935%, p<o.000l).
Figure 3C shows that a significant elevation in ALT was detected as early as
3h after
APAP treatment, and this elevation in ALT was significantly attenuated by
treatment
with ISO.
The number of HPCs in the livers of the various treatment groups was analyzed
using
flow cytometry and immunohistochemistry. As shown in Figure 3d and 3e, ISO
treatment significantly increased the number of HPCs even though injury, as
shown in
Figure 3c, was far less compared to the APAP alone group. The density of HPC
in the
smallest portal tract, was also analyzed by C1(19 positivity, as it is
reported that in APAP
induced liver injury models the density of C1(19 positive cells in the
smallest portal tract
is a more precise quantification compared to the absolute number. Figure 3f
shows that
the HPC density was significantly increased in the APAP+ISO group compared to
the
APAP alone group. To clarify the significance of P-adrenoceptor signalling in
this
model, the P-adrenoceptor antagonist PRL was used. Figure 3g shows that PRL
treatment markedly increased injury and resulted in reduced numbers of HPCs.
Example 4 - Expanding Hepatic progenitor cells are the main source of Wnt
The inventors then decided to determine how ISO treatment protects the liver
from
APAP induced injury. In order to do this, the canonical Wnt pathway was
investigated.
Canonical Wnt signalling is reported to be hepatoprotective against APAP
induced liver
injury in addition to its role in HPC proliferation.
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Mice were initially administered APAP at 500mg/kg intraperitoneally. This
resulted in
a significant number of deaths, and was reduced by ISO treatment. Therefore,
the dose
of APAP was reduced to 375mg/kg. ih after administration with APAP, mice were
treated with either ISO or PBS vehicle. As shown in Figure 4a, Western
blotting showed
that fl-catenin expression was significantly increased in the livers of ISO
treated mice
compared to those treated with APAP alone and controls at 24h after injection.
Immunohistochemistry using activated fl-catenin antibody also showed strong 13-
catenin staining in the livers of APAP+ISO treated groups. Analysis of 16
known various
Wnt ligands in the APAP and APAP+ISO treated livers showed that Wnt6, Wntioa,
Wntii and Wnti6 were upregulated in the APAP alone and APAP+ISO groups, with
Wnt6 showing significantly higher expression in the APAP+ISO group compared to
APAP only group, see Figure 4c. At this time, significant HPCs expansion was
also
detected in the livers of APAP+ISO mice. Importantly, Wnt 6, ma, n and 16 were
significantly upregulated in APAP and APAP+ISO group at 2h after APAP
administration. Among these Wnt ligands, Wnt ma showed significantly higher
expression in the APAP+ISO group. Moreover, significant fl-catenin activation
in the
APAP+ISO group was also detected at 3h after APAP administration. These data
suggested that ISO treatment enhanced the canonical Wnt pathway.
To further define the cell types in the liver responsible for these ligand
upregulation,
hepatocytes, EpCAM positive cells, and EpCAM depleted non-parenchymal cells
were
isolated from the livers of mice treated with APAP+ISO. Wnt ligand expression
in these
fractions was then anlayzed. As shown in Figure 4d, among the Wnt ligands
which was
upregulated in vivo, the EpCAM positive cell fraction showed significant
higher Wnt6,
loa, and 16 expression compared to the other fractions. In addition, the EpCAM
positive fraction showed the highest expression of Wnti and Wnt3a. Among these
upregulated Wnt ligands, Wnti, 3a, 6 and loa are known to induce the canonical
Wnt
pathway.
To evaluate the possible influence of ISO induced Wnt upregulation on liver
cells, Wnt
expression in the various liver cell types in the presence and absence of ISO
was
anlayzed ex vivo. Wnt 6 expression was detected in isolated hepatic stellate
cells (HSC)
and Kupffer cells (KC). Culture activation significantly upregulated Wnt 6
expression in
HSC compared to freshly isolated HSC. However, ISO did not induce upregulation
of
Wnt 6 in HSC or KC. These data suggest that the major source of Wnt is HPCs.
The
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inventors also investigated several cytokines which can induce HPC
proliferation but
they could not detect any ISO specific significant elevation in these
cytokines. These
results suggested that ISO treatment increases HPC number as well as their
expression
of Wnt to subsequently activate the canonical Wnt pathway in hepatocytes and
protect
from APAP toxicity.
To further support this postulate, primary hepatocytes were extracted from
mice livers
and treated with APAP. This treatment significantly induced their death as
judged by
release of LDH. ISO pre-treatment did not protect the hepatocytes from APAP
induced
death at any dose investigated. However, recombinant Wnt3a pre-treatment
significantly protected the hepatocytes against APAP. As shown in Figure 4e,
the
conditioned media from 6o3B stimulated with ISO significantly protected the
hepatocytes, an effect reversed by recombinant DKKi. Western blotting showed
that
ISO did not increase 13-catenin expression on hepatocytes but rWnt3a and ISO
stimulated conditioned media induced increased 13-catenin expression (data not
shown). These results strongly suggested that ISO protects hepatocytes from
APAP
induced cell death not directly but through paracrine activation of the
canonical Wnt
pathway.
Example 5 - Hepatic progenitor cell expansion is hepatoprotective
The above Examples indicate that expanding HPCs are the source of Wnt and that
these
HPCs have a protective role in APAP induced liver injury. To test this
hypothesis,
Tumour associated weak inducer of apoptosis (TWEAK) was used together with
direct
HPC administration. TWEAK has been reported to specifically promote progenitor
cell
expansion in the liver with no effect on hepatocytes (Jakubowski, A., et al
2005). To
take advantage of this property, recombinant TWEAK was administered before
APAP
treatment and expansion of the endogenous HPCs.
As shown in Figure 5a and 5b, TWEAK administration induced HPC proliferation
without any evidence of hepatocyte cell death, as judged by ALT and active
caspase-3
immunostaining. The effect of TWEAK is mediated by NF-kB signaling in HPCs
(Tirnitz-Parker, J.E., et al 2010) and NF-kB p65 immunostaining has revealed a
strong
cytoplasmic and nuclear expression of the protein, especially in periportal
ductular
cells, see Figure 5c. Furthermore, expansion of HPC by TVVEAK administration
protected from APAP induced liver injury, as shown in Figures 5d, se and 5f.
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As described in Figure 5g, pooled EpCAM positive cells were from the livers of
mice
treated with APAP+ISO 2h after APAP administration were then administered to
mice
which has been treated with APAP. EpCAM positive cells injection significantly
ameliorated liver injury compared to vehicle and EpCAM depleted non-
parenchymal
cells. DKK1 treatment reversed the effect of EpCAM positive cell
administration, as
shown in Figure 5h.
Example 6- Delayed administration of isoprenaline (ISO)
The effects of ISO in combination with the current gold-standard treatment,
NAC, were
also investigated. Figure 6A shows the experimental design of the study. As
shown in
Figure 6B, administration of 150mg/kg of NAC markedly reduces hepatocyte
injury
when administered 1 hour following overdose. However, NAC did not have a
protective
role if it was administered 3hrs post APAP. Conversely, ISO markedly reduced
APAP
induced-liver injury even at 3hrs post APAP.
Example 7- Effect of the ai-adrenoceptor Phenylephrine agonist on APAP induced
liver injury
To determine if the a1-adrenoceptor agonist, phenylephrine, induces effects
similar to
isoprenaline, the protocol of Example 16 (above) was repeated with ISO.
APAP was administered at 375mg/kg and either phenylepherine (PE) or PBS
vehicle
were given ih after APAP. As shown in Figure 7a, APAP alone induced
substantial liver
injury reflected by increased ALT (3500 750). This effect was moderately
reduced by
PE 3mg (2000 1200) and significantly reduced by PE lomg (450 200, p<0.005).
To investigate the pathways through which PE protects from APAP induced liver
injury,
6o3B cells were cultured in the presence and absence of PE with ISO. As shown
in
Figure 7B, it was found that PE, at lopM to io[tM, induced moderate but
significant
proliferation of 6o3B cells.
References
Hiramoto, T., Ihara, Y. & Watanabe, Y. alpha-i Adrenergic receptors
stimulation
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